Parallel 22: Pediatric Liver Diseases: Models, Modifiers, Mechanisms, and Markers
Authors: Mladen Yovchev, Fadi Jaber, Zhonglei Lu, Shachi Patel, Joseph Locker, Leslie Rogler, John Murray, Mar- Iana Dabeva, Liang Zhu, David Shafritz, Kemal Akat, Youngmin Lee, Marie-Luise Berres, Swan Thung, Meena Bansal, Thomas Tuschl, Scott Friedman, Smith Kline, Hoffman-La Roche, Marie-Luise Hong, Swan Berres, Meena Thung, Tatsuki Mizuochi, Pranavkumar Shivakumar, Reena Mourya, Stephanie Walters, Bryan Donnelly, Shiva Shanmukhappa, Jorge Bezerra, Ronald Sokol, Karim Kasmi, Padade Vue, Aimee Anderson, Michael Devereaux, Natarajan Balasubramaniyan, Frederick Suchy, James Hamilton, Lahari Koganti, James Potter, Abigael Muchenditsi, David Huso, Esteban Mezey, Svetlana Lut- Senko
Journal: Hepatology (2014)

Background: MiRNAs are abundant ~22-nt non-coding RNAs that regulate hundreds of gene targets in a sequence-and context-specific manner.While miRNAs have been described in hepatic stellate cells (HSCs), only relative changes in miRNA expression have been reported and there is no genome widemap of miRNA targets.Theoretical algorithms to predict miRNA binding-sites, while helpful, do not reflect context-dependent, real-time miRNA binding to target sites.In contrast, miRNA binding-sites in living cells can be directly analyzed using the PAR-CLIP method (photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation) (Hafner et al, 2010, Cell).Our aim was to identify miRNAs targets and target-sites on a genome-wide scale in HSCs by Ago2 PAR-CLIP.Methods: Primary HSCs were isolated from human livers, culture activated by expanding on polystyrene cell culture dishes, and the purity confirmed by FACS.A barcoded small RNA-sequencing (miRNAseq) protocol developed in our laboratory was used to quantify HSC miRNA expression, which was combined with RNAseq of polyA-RNA using the Illumina TruSeq platform to analyze target gene expression.PAR-CLIP was performed using a mouse monoclonal human anti-Ago2 antibody (clone 9E8.2).Results: There was a consistent miRNA profile across 5 biological replicates of primary HSCs, clearly distinct from primary human hepatocytes, which dominated whole liver miRNA expression.55 unique miRNAs organized in 21 cistrons, and representing 28 miRNA families constituted 90% of the sequencing reads.The top 5 most abundant miRNA cistrons were mir-21(1), mir-98(13), mir-23a(6), mir-29a(4), and mir-22(1), respectively.We obtained 30,000 Ago2 binding-regions (clusters) by PAR-CLIP representing 8000 unique genes.The clusters were almost evenly distributed over the coding sequences and 3' UTRs, and they were enriched in motifs complementary to the seed-sequence of almost all highly expressed miRNA families identified by miRNAseq.8000 clusters contained canonical 6mer, 7mer-A1, 7mer-m8, and 8mer miRNA binding-sites for these highly expressed miRNAs.Among others, we identified over hundred miR-21 binding-sites, including, but not limited to genes involved in TGF-β1 signaling or tissue fibrosis.Conclusion: Using RNAseq and PAR-CLIP we have identified thousands of bona fide miRNA binding sites in primary human HSCs at single-nucleotide resolution, uncovering both known and novel miRNA targets.This comprehensive catalogue of HSC miRNAs provides fertile directions for clarifying regulatory events and identifying novel therapeutic targets.